Current tumor treatments include radiotherapy, chemotherapy, surgical resection, hormone therapy, or a combination of these treatments. Precise control of protein turnover is essential to cellular survival. In eukaryotes, the majority of protein degradation occurs through the UPP, which consists of the ubiquitin-conjugating system and the proteasome (Yamasaki, L. and Pagano, M. Curr. Opin. Cell Biol. 2004, 16, 623-628. Ciechanover, A.; Orian, A.; Schwartz, A. L. J Cell Biochem Suppl 2000, 34, 40-51; Ciechanover, A. Cell 1994, 79, 13-21; Hochstrasser, M. Curr. Opin. Cell Biol. 1995, 7, 215-223; Coux, O.; Tanaka, K.; Goldberg, A. L. Annu Rev Biochem 1996, 65, 801-847; Baumeister, W., et al. Cell 1998, 92, 367-380; Murata, S., et al. Nat. Rev. Mol. 2009, 10, 104-115). The proteasome is a massive multicatalytic protease comprising a 20S multisubunit structure which is capped by the 19S regulatory complex at each end, forming the core of the 26S proteasome, the major extralysosomal mediator of protein degradation (Groll, M., et al. Nature (London) 1997, 386, 463-471; Adams, J. Nat. Rev. Cancer 2004, 4, 349-360). The three main catalytic activities of the proteasome; peptidylglutamyl peptide hydrolysing (PGPH), trypsin-like (T-L), and chymotrypsin-like (CT-L), are mediated by three distinct catalytic β-1, β-2, and β-5 subunits respectively (Groll, M.; Bekers, C. R.; Ploegh, H. L.; Ovaa, H. Structure, 2006, 14, 451-456). The proteasome is responsible for degrading a large number of cellular proteins (Lowe J., et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science 1995; 268:533-9). The eukaryotic proteasome possesses at least three distinct activities: chymotrypsin-like (cleavage after hydrophobic residues), trypsin-like (cleavage after basic residues), and caspase-like (cleavage after acidic residues). These target proteins are first tagged with ubiquitin in order to be degraded by the proteasome.
Ubiquitination is mediated by the sequential action of an E1 Ub-activating enzyme, an E2 Ub-conjugating enzyme, and an E3 Ub-ligase. Once Ub-tagged, proteins bind to subunits in the 19S regulatory cap of the proteasome, where they are deubiquitinated and unfolded in an energy dependent manner. These are then fed into the catalytic inner chamber of the 20S complex, which generates peptides of 3-22 amino acids in size (Vorhees, et al., The proteasome as a target for cancer therapy. Clin Can Res. 2003 Dec. 15; 9: 6316-6325).
The ATP-dependent ubiquitin-proteosome pathway is responsible for the controlled degradation of proteins in eukaryotic cells (Hochstrasser, Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr. Opin. Cell Biol. 1995, 7, 215-23; Yamasaki and Pagano, Cell cycle, proteolysis and cancer. Curr. Opin. Cell Biol. 2004, 16, 623-628; Coux, et al. Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem 1996, 65, 801-47; Ciechanover, et al. The ubiquitin-mediated proteolytic pathway: mode of action and clinical implications. J. Cell. Biochem. 2000, 40-51; Ciechanover, The ubiquitin-proteasome proteolytic pathway. Cell (Cambridge, Mass.) 1994, 79, 13-21; Baumeister, et al. The proteasome: paradigm of a self-compartmentalizing protease. Cell 1998, 92, 367-80). The 26S proteosome is a multifunctional complex, consisting of a 19S regulatory particle (RP) and a 20S core particle (CP) (Groll, et al. Structure of 20S proteasome from yeast at 2.4.ANG. resolution. Nature (London) 1997, 386, 463-471). The three main catalytic activities of the proteasome; peptidylglutamyl peptide hydrolysing (PGPH), trypsin-like (T-L), and chymotrypsin-like (CT-L), are mediated by three distinct catalytic β-1, β-2, and β-5 subunits respectively (Groll and Huber, Inhibitors of the eukaryotic 20S proteasome core particle: a structural approach. Biochim. Biophys. Acta, Mol. Cell. Res. 2004, 1695, 33-44).
In a broad range of cell culture models, proteasome inhibitors rapidly induce tumor cell apoptosis, selectively activating the cell death program in oncogene-transformed, but not normal or untransformed cells, and are able to trigger apoptotic death in human cancer cells that are resistant to various anticancer agents (Adams J. Preclinical and clinical evaluation of proteasome inhibitor PS-341 for the treatment of cancer. Curr Opin Chem Biol 2002; 6:493-500; Dou Q., Goldfarb R. Evaluation of the proteasome inhibitor MLN-341 (PS-341). IDrugs 2002; 5:828-834). Inhibition of the chymotrypsin-like, but not the trypsin-like, activity has been found to be associated with induction of tumor cell apoptosis.
Apoptosis is a highly conserved cellular suicide program in multicellular organisms from worms to humans. This cellular death program serves as a means to maintain multicellular organisms by discarding damaged and undesirable cells. Faulty execution of apoptosis, including either excessive cell death or insufficient cell death, is a factor in many disease states including AIDS and cancer (Jacobson M., et al. Programmed cell death in animal development. Cell 1997; 88:347-54; Song Z., Steller H. Death by design: mechanism and control of apoptosis. Trends Cell Biol 1999; 9:M49-52). Apoptosis features several distinct events and morphological changes, such as loss of the mitochondrial membrane potential, proteolytic dismantling of cellular components, DNA fragmentation, and cellular condensation into apoptotic bodies that are removed by phagocytes (Green D., Reed J. Mitochondria and apoptosis. Science 1998; 281:1309-12; Earnshaw W., et al. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 1999; 68:383-424; Bratton S., et al. Protein complexes activate distinct caspase cascades in death receptor and stress-induced apoptosis. Exp Cell Res 2000; 256:27-33; Wyllie A., et al. Cell death: the significance of apoptosis. Int Rev Cytol 1980; 68:251-306). As a distinct series of cellular pathways, apoptosis potentially offers unique targets for chemotherapeutic intervention. It has been suggested that cancer cells are more sensitive to several apoptosis-inducing stimuli than normal cells, including proteasome inhibitors and those affecting cellular division (Adams J. Potential for proteasome inhibition in the treatment of cancer. Drug Discov Today 2003; 8:307-15; Dou Q., Li B. Proteasome inhibitors as potential novel anticancer agents. Drug Resist Update 1999; 2:215-223; Almond J B, Cohen G M. The proteasome: a novel target for cancer chemotherapy. Leukemia 2002; 16:433-43; Goldberg A L. Functions of the proteasome: the lysis at the end of the tunnel. Science 1995; 268:522-3; Dou Q., et al. Interruption of tumor cell cycle progression through proteasome inhibition: implications for cancer therapy. In Progress in Cell Cycle Research. Meijer L, Jezequel A, Roberge M, (eds.) Life in Progress Editions, Roscoff, 2003; pp. 441-446). Several regulatory proteins involved in cell cycle and apoptosis processes, such as cyclins, bcl-2 family members, and p53, are degraded by the ubiquitin-proteasome pathway (An B., et al. Novel dipeptidyl proteasome inhibitors overcome Bcl-2 protective function and selectively accumulate the cyclin-dependent kinase inhibitor p27 and induce apoptosis in transformed, but not normal, human fibroblasts. Cell Death Differ 1998; 5:1062-75; Lopes U., et al. p53-dependent induction of apoptosis by proteasome inhibitors. J Biol Chem 1997; 272:12893-6).
Owing to the central role of proteosome in maintaining homeostasis and hence its key position in many cellular processes, the development of proteasome inhibitors for CT-L activity has been the subject of considerable interest in the treatment of cancer due to its critical role in the degradation of apoptotic and tumor suppressor proteins (Borissenko and Groll, 20S Proteasome and Its Inhibitors: Crystallographic Knowledge for Drug Development. Chem. Rev. (Washington, D.C., U.S.) 2007, 107, 687-717; Genin, et al. Proteasome inhibitors: recent advances and new perspectives in medicinal chemistry. Curr. Top. Med. Chem. (Sharjah, United Arab Emirates) 2010, 10, 232-256). The antitumor activity of proteasome inhibitors has been confirmed by the results of bortezomib, a potent and selective dipeptidyl boronic acid proteasome inhibitor (Sunwoo J., et al. Novel proteasome inhibitor PS-341 inhibits activation of nuclear factor-kappa B, cell survival, tumor growth, and angiogenesis in squamous cell carcinoma. Clin Cancer Res 2001; 7:1419-28) that selectively inhibits the CT-L activity of 20S proteasome (Jung, L., et al. Oncology 2004, 18, 4-13; Lara, P. N., et al. Semin. Oncol. 2004, 31, 40-46. Adams, J. Semin. Oncol. 2001, 28, 613-619).
The proteasome inhibitors currently in the clinic are derived from 3 structural classes, seen in FIG. 1: In the first class, Bortezomib, which is a dipeptide boronic acid, was the first clinically approved proteasome inhibitor (Groll, et al. Snapshots of the Fluorosalinosporamide/20S Complex Offer Mechanistic Insights for Fine Tuning Proteasome Inhibition. J. Med. Chem. 2009, 52, 5420-5428; Groll, et al. Crystal structure of epoxomicin:20S proteasome reveals a molecular basis for selectivity of a′,b′-epoxyketone proteasome inhibitors. J. Am. Chem. Soc. 2000, 122, 1237-1238). Similar to Bortezomib, MLN9708 (Kupperman, et al. Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer. Cancer Res 2010, 70, 1970-80; Kupperman, et al. Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer. [Erratum to document cited in CA152:517050]. Cancer Res. 2010, 70, 3853; Lawrence, et al. Building on bortezomib: second-generation proteasome inhibitors as anti-cancer therapy. Drug Discov Today 2010, 15, 243-9) (a modified dipeptidyl boronic acid which hydrolyses immediately in plasma) and CEP-18770 (Dorsey, et al. CEP-18770: Discovery of a Potent, Selective and Orally Active Proteasome Inhibitor for the Treatment of Cancer. Frontiers in CNS and Oncology Medicinal Chemistry, ACS-EFMC, Siena, Italy, Oct. 7-9 2007, COMC-027; Piva, et al. CEP-18770: a novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib. Blood 2008, 111, 2765-2775; Sterz, et al. The potential of proteasome inhibitors in cancer therapy. Expert Opin. Invest. Drugs 2008, 17, 879-895) are also boronic acid derivatives. The second class includes β-lactone salinosporamide A (Fuchs, Proteasome inhibition as a therapeutic strategy in patients with multiple myeloma. Mult. Myeloma 2009, 101-125; Lam, et al. From natural products to clinical trials: NPI-0052 (salinosporamide A), a marine actinomycete-derived anticancer agent. Nat. Prod. Chem. Drug Discovery 2010, 355-373) (represented by NPI-0052) is a marine microbial natural product. The third class includes tetrapeptide epoxyketone carfilzomib (Zhou, et al. Design and Synthesis of an Orally Bioavailable and Selective Peptide Epoxyketone Proteasome Inhibitor (PR-047). J. Med. Chem. 2009, 52, 3028-3038), which is related to the natural product epoxomicin. Each inhibitor class reacts with the proteasome N-terminal threonine active sites by a distinct mechanism. Peptide boronic acids (Bortezomib, MLN9708 and CEP-18770) form a slowly reversible tetrahedral adduct with the OH group of the catalytic Thr-1 (Groll, et al. Crystal Structure of the Boronic Acid-Based Proteasome Inhibitor Bortezomib in Complex with the Yeast 20S Proteasome. Structure (Cambridge, Mass., U.S.) 2006, 14, 451-456). For the β-lactone NPI-0052, attack of the lactone ring by catalytic Thr-123 forms an ester bond (that undergo intramolecular rearrangement) which makes this compound an irreversible inhibitor. The epoxyketone (Groll, et al. Crystal structure of epoxomicin:20S proteasome reveals a molecular basis for selectivity of a′,b′-epoxyketone proteasome inhibitors. J. Am. Chem. Soc. 2000, 122, 1237-1238) moiety of Carfilzomib reacts with the OH and the α-amino group of Thr-1 to form 2 covalent bonds, making the inhibition also irreversible.
Proteasome inhibitors are classified as reversible or irreversible inhibitors according to their chemical structure and mechanism of inhibition. Irreversible/covalent and slow reversible inhibitors as described above possess a chemically reactive group that bind to proteasome covalently; whereas non-covalent and rapidly reversible inhibitors inhibit the proteasome through a network of interactions (hydrophobic, hydrogen bonds, electrostatic and/or van der Waals). Examples of reversible proteosome inhibitors include Ritonavir (Schmidtke, et al. How an inhibitor of the HIV-I protease modulates proteasome activity. J. Biol. Chem. 1999, 274, 35734-35740), several benzylstatine derivatives (Furet, et al. Structure-Based optimization of 2-aminobenzylstatine derivatives: potent and selective inhibitors of the chymotrypsin-Like activity of the human 20S proteasome. Bioorg. Med. Chem. Lett. 2002, 12, 1331-1334), 5-trimethoxy-L-phenylalanine derivatives (Furet, et al. Entry into a New Class of Potent Proteasome Inhibitors Having High Antiproliferative Activity by Structure-Based Design. J. Med. Chem. 2004, 47, 4810-4813), lipopeptides (Basse, et al. Development of lipopeptides for inhibiting 20S proteasomes. Bioorg. Med. Chem. Lett. 2006, 16, 3277-3281), TMC-95A derivatives (Kohno, et al. Structures of TMC-95A-D: Novel proteasome inhibitors from Apiospora montagnei Sacc. TC 1093. J. Org. Chem. 2000, 65, 990-995) and fluorinated pseudopeptides (Formicola, et al. Novel fluorinated pseudopeptides as proteasome inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 83-86). Since non-covalent inhibitors do not have a reactive moiety, which is often associated with metabolic instability, poor specificity, and excessive reactivity, they have the advantage of exerting fewer side effects over the covalent and irreversible ones. It has been shown that the proteasome activity recovers at the same rate with irreversible inhibitors as with covalent and slowly reversible inhibitors, presumably due de novo proteasome synthesis. The clinical advantages/benefits of non-covalent and reversible proteasome inhibitors in cancer treatment are not well understood. We have been actively engaged in the discovery of novel proteasome inhibitors (Lawrence, et al. Synthesis and biological evaluation of naphthoquinone analogs as a novel class of proteasome inhibitors. Bioorg. Med. Chem. 2010, 18, 5576-5592; Ge, et al. Discovery and Synthesis of Hydronaphthoquinones as Novel Proteasome Inhibitors. J. Med. Chem., 2012 Mar. 8; 55(5):1978-98. Epub 2012 Feb. 14).
The potent proteasome inhibitors reported to date have been developed as aldehydes, boronates, vinylsulfones and expoxyketones and these compounds function through covalent modification of the N-terminal threonine residue of β-5 subunit. However, toxicity and tumor cell resistance against Bortezomib, as well as other proteasome inhibitors, demand the development of improved and selective proteasome inhibitors.